Lupus is a multisystem autoimmune disease where many organs, including the kidney, can be affected. It is a chronic inflammatory disease the pathophysiology of which is manifested by the production of autoantibodies directed against multiple self-antigens, particularly those of nuclear origin. This dysregulation of the immune system results in a loss of self-tolerance, and is mediated by both T and B cells. (Reddy et al, Arthritis Research & Therapy, 2008, 10:R127; and references therein). There are very few medications approved for the treatment of lupus (Francis & Perl, 2009; Mok, 2010). These include: Prednisone (flare up and maintenance treatment), hydroxychloroquine (discoid lupus and SLE), aspirin (arthritis and pleurisy), triamcinolone hexacetonide (discoid lupus), and most recently Benlysta (SLE). In addition, several other agents are regularly prescribed including azathioprine (as a corticosteroid sparing agent), and in more aggressive regimens corticosteroids in combination with variations of cyclophosphamide, mycophenolate mofetil, or the calcineurin inhibitors such as cyclosporine and tacrolimus (Mok, 2010). For patients who are intolerant or refractory to the above listed agents, several biological agents have been utilised including intravenous immunoglobulin and the B cell depleting agent rituximab, although safety concerns have been raised about the latter through a potential link to progressive multifocal leukoencephalopathy infection.
A number of new agents are in development for the treatment of lupus. These include studies to find safer ways to use the immunosuppressive therapies described above (azathioprine, cyclophosphamide, mycophenolate mofetil), and several B cell targeting antibody therapies which exhibit potent effects but significant adverse events (for reviews see, Mok, 2010; Francis & Perl, 2009).
It has been shown that mTOR (mammalian target of rapamycin) activity is upregulated in the T cells of autoimmune patients including lupus and multiple sclerosis (MS) (Fernandez et al., 2009a,b), and that inhibition of mTORC1 by rapamycin and its analogs inhibits antigen-induced IL-2 driven T and B cell proliferation. Moreover, the activity of rapamycin and its analogues do not block proliferation of all T cell subtypes, and actually induce selective expansion of regulatory T cells (Tregs) which are important in maintaining immune self-tolerance (Donia et al., 2009; Esposito et al., 2010).
Abnormal T cell activation in SLE is linked to sustained elevation of the mitochondrial transmembrane potential, which is in turn controlled by a series of metabolic and stress related inputs (Perl et al., 2004; Fernandez et al, 2009; Fernandez & Perl, 2009). mTOR is a sensor for these inputs and as a consequence elevated mTOR signalling is observed in lupus T cells, an effect which is normalised by treatment with rapamycin (Perl et al., 2004; Fernandez et al., 2009). Moreover, two independent studies have identified a network of genes which are dysregulated in lupus/nephritis associated disease. There is a strong correlation between the abnormal transcription of these gene networks and mTOR signalling, and treatment with rapamycin returns the levels of gene transcription to asymptomatic levels. (Reddy et al., 2008; Wu et al., 2007).
Rapamycin is effective when dosed orally in preclinical mouse models of lupus. For example using the New Zealand Black White NZBW/F1 mouse model of lupus nephritis rapamycin significantly improved the clinical course of lupus nephritis (Stylianou et al., 2010; Liu et al., 2008). In the study by Liu et al (2008), treatment with rapamycin significantly decreased albuminuria, improved survival, diminished splenomegaly, preserved renal function and reduced serum anti-dsDNA antibody levels. Kidney sections from saline-treated mice revealed marked mesangial proliferation, tubular dilation with intra-tubular protein cast deposition and leukocytic infiltration of the interstitium. The rapamycin-treated mice, in contrast, had relatively mild histological changes in their kidneys. Rapamycin treatment also significantly reduced the amount of immune complex deposition in the glomeruli, suppressed the interstitial infiltration by T-cells, B-cells and macrophages as well down-regulated the intra-renal expression of RANTES. Stylianou et al (2010) found that in untreated mice, as opposed to healthy controls, Akt and mTOR were over-expressed and phosphorylated at key activating residue and rapamycin also prolonged survival, maintained normal renal function, normalized proteinuria, restored nephrin and podocin levels, reduced anti-dsDNA titres, ameliorated histological lesions, and reduced Akt and mTOR glomerular expression activation. Additional successful examples of using rapamycin in the NZBW/F1 mouse have also been published (Alperovich et al., 2007; Liu et al., 2008b). Rapamycin has also been shown to prolong survival and to reduce inflammatory changes in several organs, including the kidneys, in the MRL/Ipr model of murine SLE (Warner et al., 1994).
Rapamycin has been studied in humans as part of a clinical trial of nine patients with SLE who had been treated unsuccessfully with other immunosuppressive medications (Fernandez et al., 2006). Rapamycin was well tolerated and proved effective for the reduction and control of disease activity in all 9 patients. Disease activity by BILAG (British isles Lupus Assessment Group) score and SLEDAI (SLE Disease Activity Index) and concurrent prednisone dosage at the time of rapamycin initiation and the last follow up visit (after 6-48 months of treatment) were reported. The BILAG disease flare index was reduced in 7 patients, unchanged in 1 patient, and increased in 1 patient. In the latter patient the increase in the BILAG score (which is considered to be a highly sensitive instrument for detecting disease flares) was due to transient arthralgias during the last observation period, which did not require adjustment of the prednisone dosage. At the last follow up, the mean±SEM reduction in the BILAG score in the 9 patients compared with pretreatment was 1.93±0.9 (P=0.0218). The SLEDAI was reduced by 5.3±0.8 (range 2-8) (P=0.00002). After treatment with rapamycin, the mean±SEM reduction in the daily dosage of prednisone in the 7 prescribed patients was 26.4±6.7 mg (P=0.0062). Three of the patients had cyclophosphamide-treated lupus nephritis. In all 3, the nephritis remained in remission throughout the period of rapamycin treatment, with normal serum creatinine levels and urinary protein levels of <300 mg/24 hours.
The clinical features of 7 control SLE patients not treated with rapamycin were measured. The disease activity scores of these patients were higher than those of patients who had received 6-48 months of rapamycin treatment (mean BILAG score 5.00 versus 2.11 [P=0.02]; mean SLEDAI score 3.14 versus 1.55 [P=0.11]). These observations are consistent with the notion that rapamycin treatment is beneficial in SLE. T cells from 7 healthy controls, 7 SLE controls, and 6 rapamycin-treated SLE patients were used for studies of Ca2+ signaling and mitochondrial transmembrane potential. While mitochondrial hyperpolarization (MHP) persisted, baseline [Ca2+]c, and [Ca2+]m and T cell activation-induced rapid Ca2+ fluxing were normalized in rapamycin-treated patients. T cells from SLE patients not receiving rapamycin showed significantly elevated Ca2+ levels at each time point, with P values versus levels in healthy controls ranging between 0.0008 at time 0 to 0.023 at 16 minutes. In contrast, the level of CD3/CD28-induced Ca2+ fluxing in T cells from rapamycin-treated SLE patients was not significantly different from that in cells from healthy donors.
In summary, rapamycin has shown positive effects in murine lupus, and the findings in a human clinical study of 9 rapamycin-treated patients indicated that rapamycin can effectively control disease activity in SLE. Arthritis improved in all 9 patients, and cyclophosphamide treated nephritis in 3 patients remained in remission during rapamycin treatment. The single daily oral administration and small size of the pill was liked and well-tolerated by all patients. None of the patients discontinued the drug due to lack of efficacy or adverse effects.
Patients with lupus are at high risk of atherosclerosis (Gorman & Isenberg, 2004). Indeed, various estimates suggest that up to 30% of deaths in lupus patients may be due to coronary artery disease (Aranow & Ginzler, 2000; Petri et al., (1992); Gorman & Isenberg, 2004). It is known that rapamycin and other clinically used rapamycin analogues cause an elevation in circulating lipid/triglycerides and cholesterol levels in human patients (Morisett et al., 2002). Given the well-established link between elevated blood levels of lipid/triglycerides and cholesterol and atherosclerosis/cardiovascular disease, rapamycin analogues which are similarly effective as rapamycin as an mTOR inhibitor, but which have a significantly lesser effect on the elevation of lipid/triglycerides and cholesterol levels would be extremely useful for the treatment of lupus and other diseases in which mTOR inhibition may be effective. The range of diseases where rapamycin, or improved analogues, may be effective as pharmacological agents includes, but is not limited to, lupus, multiple sclerosis, Parkinson's disease, Huntingdon's disease, Alzheimer's disease. A recent review of neurological indications where rapamycin or rapamycin analogues may be effective treatments has been published (Bove et al., 2011).
Multiple sclerosis (MS) is a chronic autoimmune disorder of the central nervous system (CNS) that is characterized by inflammation leading to astrogliosis, demyelination, and loss of oligodendrocytes and neurons (Brinkmann et al., 2010; Compston & Coles, 2002). MS is the leading cause of neurological disability in young and middle-aged adults, affecting an estimated 2.5 million individuals worldwide (Multiple Sclerosis International Federation. Atlas of MS Database. Multiple Sclerosis International Federation website [online], http://www.atlasofms.org/index.aspx (2008)). The prevalence is greatest in Caucasians, with high prevalence rates reported in Europe, Canada, USA, Australia, New Zealand and northern Asia (Rosati, 2001; Noseworthy et al., 2000). Most patients are diagnosed between the ages of 20 and 40 years (in a 2:1 female to male ratio) (Compston & Coles, 2002). At diagnosis, ˜85% of patients have relapsing-remitting MS (RRMS), which is characterized by recurrent acute exacerbations (relapses) of neurological dysfunction, followed by recovery. A substantial proportion (42-57%) of relapses may result in incomplete recovery of function and lead to permanent disability and impairment (Lublin et al., 2003). Within 6-10 years of disease onset, 30-40% of patients with RRMS have progressed to secondary progressive MS (Weinshenker et al., 1989), in which a less inflammatory and more neurodegenerative course of disease seems to take precedence. Secondary progressive MS presents with steady progression in disability, with or without superimposed relapses.
Treatment strategies for MS usually involve the management of symptoms and the use of disease-modifying drugs to reduce the frequency of relapses and to slow the progression of disability. Established first-line therapies—interferon-β (IFN-β) products and glatiramer acetate (Copaxone; Teva)—provide ˜30-35% reduction in the relapse rate compared with placebo over 2 years (PRISMS Study Group, 1998; The IFNB Multiple Sclerosis Study Group, 1993; Jacobs et al., 1996; Johnson et al., 1995). IFN-β1a has also been shown to reduce the progression of disability in patients with RRMS (Goodin et al., 2002). These agents are administered by injections (with dosing schedules ranging from daily subcutaneous injections to weekly intramuscular injections), and may affect the immune system on several levels. More frequent side effects include influenza-like symptoms and injection-site reactions, which can affect tolerability and compliance (Patti, 2010). Less commonly reported adverse events for IFN-β therapies include liver dysfunction and cytopaenias (Rice, et al., 2001).
A more recently approved therapy, natalizumab (Tysabri; Elan/Biogen-Idec), is a humanized monoclonal antibody specific for the α4 subunit of the integrin α4βI (also known as very late antigen 4) on lymphocytes (Steinman, 2005; Putzki et al., 2010). It is administered through intravenous infusions every 4 weeks and seems to offer enhanced efficacy compared with other approved products (Putzki et al., 2010). However, natalizumab has been associated with hypersensitivity reactions and with progressive multifocal leukoencephalopathy, a rare but seriously disabling or fatal infectious demyelinating disease of the brain (Steinman, 2005). Another product, the cytostatic agent mitoxantrone (for which the cellular target has not been identified), is approved for use in severe forms of relapsing MS. However, cumulative dose-related cardiac toxicity and a risk of secondary leukaemia limit the total amount that can be administered (Kingwell, et al., 2010). Because of their safety profiles, natalizumab and mitoxantrone are currently used only as second- and third-line treatments. Drugs under development for MS include the monoclonal antibodies rituximab, ocrelizumab and ofatumumab, which target CD20 to deplete B cells, as well as alemtuzumab (Campath-1H), which targets CD52 to deplete T and B cells and some monocyte-derived dendritic cells (Buttmann, 2010). Also in development are small molecules, including the oral agents cladribine (a cytotoxic adenosine deaminase-resistant purine nucleoside), fumarate (an activator of the nuclear factor E2-related factor 2 transcriptional pathway), laquinimod (the cellular target of which has not been identified), and teriflunomide (a cytostatic inhibitor of dihydroorotate dehydrogenase, which catalyses the rate-limiting step in the de novo synthesis of pyrimidines). All these agents target lymphocytes as well as other cells with the aim of inhibiting the immune-system-mediated attack on the CNS (Niino, & Sasaki, 2010). The sphingosine 1-phosphate (S1P) receptor modulator fingolimod (FTY720/Gilenya; Novartis) was the first oral treatment for RRMS approved by the US FDA (Brinkmann et al., 2010). Although the trials so far have shown fingolimod to be well tolerated, the side effects that have occurred include headache, upper respiratory tract infection, shortness of breath, diarrhea and nausea. In addition, increased levels of liver enzymes and blood pressure have been observed although these are generally mild. In the TRANSFORMS clinical trial (Trial Assessing Injectable Interferonvs. FTY720 Oral in RRMS; ClinicalTrials.gov number, NCT00340834), two deaths resulting from herpes virus infections occurred in patients taking the higher dose of fingolimod (see Garber, 2008). Other aspects of the treatments these two patients received may have contributed, but a role for fingolimod cannot be excluded given its immunomodulatory action, which could lead to an increased risk of infections.
Given the limitations of currently available therapies, the development of oral MS treatments that might offer more effective and more convenient treatment has been the focus of considerable drug discovery and development efforts in recent years.
The efficacy of rapamycin and its analogues in MS is likely attributable to a combination of their neuroprotective activity due to immunophilin/neurophilin inhibition, and the anti-inflammatory/immunosuppressive activity driven by selective mTORC1 inhibition, and remylinating properties which may be driven through both mechanisms. Rapamycin and its analogues are immunosuppressive, anti-inflammatory molecules which modulate T cell proliferation through their ability to inhibit mTOR complex 1 (mTORC1) after first binding the immunophilin FKBP12. mTOR activity is upregulated in individuals suffering from autoimmune disorders including MS and Lupus (Fernandez et al., 2009a,b), and inhibition of mTORC1 by rapamycin and its analogues inhibits antigen-induced IL-2 driven T (and B) cell proliferation. Rapamycin and its analogues do not block proliferation of all T cell subtypes, and actually induce selective expansion of regulatory T cells (Tregs) which are important in maintaining immune self-tolerance (Donia et al, 2009; Esposito et al, 2010).
Rapamycin and its analogues bind tightly to the FK506-binding protein (FKBP) family of immunophilins (Cao & Konsolaki, 2011; Gerard et al., 2011). The FKBP family consists of proteins with a variety of protein-protein interaction domains and versatile cellular functions (Kang et al., 2008). All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. Binding of rapamycin or analogues to this domain inhibits their PPIase activity while mediating immune suppression through inhibition of mTOR. The larger members, FKBP51 and FKBP52, interact with Hsp90 and exhibit chaperone activity that is shown to regulate steroid hormone signalling. From these studies it is clear that FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous system. Consistent with this expression, FKBPs have been implicated with both neuroprotection and neurodegeneration (Cao & Konsolaki, 2011; Gerard et al., 2011; Bove et al., 2011; Kang et al., 2008). Rapamycin is a nM inhibitor of the PPIase activity of several neurophilins including FKBP12 and FKBP52, and binding to these proteins has been shown to contribute to their neuroprotective effects (Ruan et al, 2008). FKBP52 binds Tau, and Tau protein overexpression is linked to inhibition of neurite outgrowth and neuroprotection (Chambraud et al., 2010). FKBP52 controls chemotropic guidance of neural growth cones via regulation of TRPC1 channel opening (Shim et al., 2009). These data provide a link for the neurite outgrowth promoting, axonal regeneration and neuroprotective effects observed for FKBP52 knockdown/inhibition. FKBP12 has been proposed many times as the major mediator of the neuroprotective effects of immunophilins, for example FK506 protection against oxygen-glucose deprivation induced damage was not present when an anti-FKBP12 antibody was added (Labrande et al., 2006), expression of FKBP12 is increased in the brain of patients with Parkinson's Disease, Alzheimer's disease and some forms of dementia (Avramut et al., 2002). It has also been implicated as the most potent enhancer of α-synuclein aggregation (Gerard et al., 2010, Deleersnijder et al., 2011).
Rapamycin has been shown effective in preclinical experimental models of MS. For example, the effect of rapamycin administration to SJL/j mice affected by PLP139-151-induced relapsing-remitting experimental autoimmune encephalomyelitis (RR EAE) has been reported (Esposito et al., 2010). Oral or intraperitoneal treatment at the peak of disease or at the end of the first clinical attack, dramatically ameliorated the clinical course of RR-EAE. Treatment suspension resulted in early reappearance of disease. Clinical response was associated with reduced central nervous system demyelination and axonal loss. The dual action of rapamycin on both Teff and Treg cells resulted in modulation of their ratio that closely paralleled disease course. The data showed that rapamycin inhibits RR-EAE, gave evidence for the immunological mechanisms, and indicated this compound as a potential candidate for the treatment of multiple sclerosis. In a second example (Donia et al., 2009), evaluated the effects of rapamycin on the course of protracted relapsing experimental allergic encephalomyelitis (PR-EAE) in Dark Agouti (DA) rats, which serves as a preclinical model of MS. The data showed that the oral administration of rapamycin at 3 mg/kg for 28 consecutive days significantly ameliorated the course of PR-EAE in DA rats. The rats that received the medication had significantly lower clinical cumulative scores and shorter duration of the disease than did the control rats treated with the vehicle.
The clinical utility in MS of rapamycin has been shown directly, but its close analogue and mTOR inhibitor temsirolimus has been studied up to Phase 2B, where the efficacy and safety of temsirolimus was evaluated in patients with clinically definite relapsing-remitting MS (RRMS) or secondary progressive MS with relapses (Kappos et al., 2005; Moraal et al., 2010). It was a multicentre, randomized, double-blind, placebo-controlled, phase 2 clinical trial conducted in 296 patients aged 19-57 years. Patients received oral temsirolimus (2, 4, or 8 mg,) 1×daily, or placebo, for 9 months. The primary end point was the cumulative number of new Gd-enhanced T1 lesions at 9 months on MRI. Total brain volume, number of relapses, mean EDSS scores, other MRI measures and health outcomes were secondary end-points.
Patients receiving 8 mg temsirolimus achieved significant reductions (47.8%) in the cumulative number of new Gd-enhancing T1 lesions on MRI compared with placebo (p=0.010). MRI endpoints showed a dose response, the 8 mg dose reaching statistical significance for the primary endpoint by 32 weeks (p=0.024). Brain volume data suggested a decrease in brain volume atrophy at 36 weeks in the 8 mg group compared with placebo. The 8 mg group showed a 51% reduction in number of relapses per patient vs. placebo (p=0.023). Dose-related trends in percentage of relapse-free patients and progression of disability were also noted. Serious adverse events occurred at similar frequencies across all treatment groups. Aphthous stomatitis/mouth ulceration, hyperlipidaemia, rashes, and menstrual dysfunction were reported more often in the 8 mg group vs. placebo. It was concluded that an oral, 8 mg dose of temsirolimus administered over 9 months in patients with relapsing forms of MS resulted in significant beneficial effects on the incidence of new enhancing MRI lesions and number of relapses, with an acceptable risk/benefit profile.
Rapamycin (sirolimus) (FIG. 4) is a lipophilic macrolide produced by Streptomyces hygroscopicus NRRL 5491 (Sehgal et al., 1975; Vezina et al., 1975; U.S. Pat. No. 3,929,992; U.S. Pat. No. 3,993,749) with a 1,2,3-tricarbonyl moiety linked to a pipecolic acid lactone (Paiva et al., 1991). For the purpose of this invention rapamycin is described by the numbering convention of McAlpine et al. (1991) in preference to the numbering conventions of Findlay et al. (1980) or Chemical Abstracts (11th Cumulative Index, 1982-1986 p60719CS).
Rapamycin has significant therapeutic value due to its wide spectrum of biological activities (Huang et al, 2003). The compound is a potent inhibitor of the mammalian target of rapamycin (mTOR), a serine-threonine kinase downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signalling pathway that mediates cell survival and proliferation. This inhibitory activity is gained after rapamycin binds to the immunophilin FK506 binding protein 12 (FKBP12) (Dumont, F. J. and Q. X. Su, 1995). In T cells rapamycin inhibits signalling from the IL-2 receptor and subsequent autoproliferation of the T cells resulting in immunosuppression. Rapamycin is marketed as an immunosuppressant for the treatment of organ transplant patients to prevent graft rejection (Huang et al, 2003). In addition to immunosuppression, rapamycin has found therapeutic application in cancer (Vignot et al, 2005), and has potential therapeutic use in the treatment of a number of diseases, for example, cancer, cardiovascular diseases such as restenosis, autoimmune diseases such as multiple sclerosis and lupus, rheumatoid arthritis, fungal infection and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease.
Despite its utility in a variety of disease states rapamycin has a number of major drawbacks. The most serious adverse event associated with its use is hyperlipidemia. This can lead to dose reduction and treatment withdrawal. In particular, any potential this class has in SLE is limited due to the naturally high lipid levels in these patients (Aranow & Ginzler, 2000; Petri et al., (1992); Gorman & Isenberg, 2004). It is also a substrate of cell membrane efflux pump P-glycoprotein (P-gp; LaPlante et al, 2002, Crowe et al, 1999) which pumps the compound out of the cell making the penetration of cell membranes by rapamycin poor. This causes poor absorption of the compound after dosing. In addition, since a major mechanism of multi-drug resistance of cancer cells is via cell membrane efflux pump, rapamycin is less effective against multi-drug resistance (MDR) cancer cells. Rapamycin is also extensively metabolised by cytochrome P450 enzymes (Lampen et al, 1998). Its loss at hepatic first pass is high, which contributes further to its low oral bioavailability. The role of CYP3A4 and P-gp in the low bioavailability of rapamycin has been confirmed in studies demonstrating that administration of CYP3A4 and/or P-gp inhibitors decreased the efflux of rapamycin from CYP3A4-transfected Caco-2 cells (Cummins et al, 2004) and that administration of CYP3A4 inhibitors decreased the small intestinal metabolism of rapamycin (Lampen et al, 1998). The low oral bioavailability of rapamycin causes significant inter-individual variability resulting in inconsistent therapeutic outcome and difficulty in clinical management (Kuhn et al, 2001, Crowe et al, 1999).
Therefore, there is a need for the development of novel rapamycin-like compounds that have reduced side effects, including hyperlipidaemia, are not substrates of P-gp, and that may be metabolically more stable and therefore may have improved bioavailability.
A range of synthesised rapamycin analogues using the chemically available sites of the molecule has been reported. The description of the following compounds was adapted to the numbering system of the rapamycin molecule described in FIG. 4. Chemically available sites on the molecule for derivatisation or replacement include C40 and C28 hydroxyl groups (e.g. U.S. Pat. No. 5,665,772; U.S. Pat. No. 5,362,718), C39 and C16 methoxy groups (e.g. WO 96/41807; U.S. Pat. No. 5,728,710), C32, C26 and C9 keto groups (e.g. U.S. Pat. No. 5,378,836; U.S. Pat. No. 5,138,051; U.S. Pat. No. 5,665,772). Hydrogenation at C17, C19 and/or C21, targeting the triene, resulted in retention of antifungal activity but relative loss of immunosuppression (e.g. U.S. Pat. No. 5,391,730; U.S. Pat. No. 5,023,262). Significant improvements in the stability of the molecule (e.g. formation of oximes at C32, C40 and/or C28, U.S. Pat. No. 5,563,145, U.S. Pat. No. 5,446,048), resistance to metabolic attack (e.g. U.S. Pat. No. 5,912,253), bioavailability (e.g. U.S. Pat. No. 5,221,670; U.S. Pat. No. 5,955,457; WO 98/04279) and the production of prodrugs (e.g. U.S. Pat. No. 6,015,815; U.S. Pat. No. 5,432,183) have been achieved through derivatisation.
An object of the invention is to identify a further derivative of rapamycin which retains its beneficial effects in therapy without some or all of its side effects. In addition, it is advantageous to have a molecule that has more potent FKBP12 inhibition.